Lecture 28 Flashcards
(39 cards)
1
Q
When is HCV treatment indicated and what’s the goal of treatment?
A
- When: chronic liver inflammation, presence of HCV RNA in the blood, active viral infection
- Goal: eliminated detectable RNA from the blood (viral clearance or sustained virological response (SVR) considered cured)
2
Q
First option of treatment?
A
- IFN alpha
- all early protocols were based on IFN alpha administered by injection multiple times a week, the modified or pegylated form slows elimination and is only given once a week still by injection
- Treatments for 24-48 weeks
- Severe side effects since we already produce IFN so like insane amount causing flu-like symptoms, depression, anxiety, restlessness, rashes, insomnia, loss of appetite, anemia, etc.
- only 30-40% of patients respond to peg-IFN alpha alone as in achieve SVR, not very efficient and not very tolerable
3
Q
2nd option of treatment?
A
- In comes ribavirin
- synthetic nucleoside analog, looks like guanine, (pseudobase) so mechanism is error-catastrophe, so many mistakes => eventually failure
- Side effects: also quite sever and include anemia, teratogenic, fatigue, headache, insomnia, nausea, anorexia, etc.
- broadly antiviral, not effective agains HCV when given alone only works in combination with IFN for example
4
Q
mechanism of action of ribavirin?
A
- MANY MECHANISM BUT ONLY focus on RNA mutagenesis, it is main mechanism of action that contributes, error-catastrophe, so viral mutagen through incorporation of ribavirin
5
Q
What were the effects of combining of ribavirin and IFN alpha?
A
- around 50% SVR, better than IFN alone but a variety of responses: some SVR, some went undetectable then relapsed post-treatment as soon as the drug was removed, some non-response and some had a drop in level of viral RNA but never became undectedctable
- BUT important to consider that 10-20% of patients did not even complete the treatment because side effects and complicated, IFN 1/week injection and ribavirn multiple times a days, plus it was expensive like 4000$/week and goes on for so long 24-48 weeks
- large teams were needed to help monitor/treat side effects/stimulate patients into compliance so they take meds
6
Q
Why were we finally able to develop DAAs?
A
Thanks to being able to culture infectious HCV in cell cultures
7
Q
What are the three classes of DAAs?
A
- Protease inhibitors
- NS5A inhibitors
- Polymerase inhibitors
8
Q
How was the protease inhibitor designed?
A
- NS3 cleaves the boundaries between non-structural proteins and when got to the NS5A/5B boundary stalled a bit on a peptide on active site after cleavage, so they used the sequence of this peptide to inhibit the catalytic site of NS3, it was possible to purify and perform enzymatic assays like for NS3 protease
- The two obtained were boceprevir and telaprevir, made plenty of analogs of the peptide and selected for the ones that actually worked inhibit NS3 using the enzymatic assays
9
Q
Use of protease inhibitors?
A
- Boceprevir and telaprevir FDA approved in 2011
- used in combination with IFNalpha and RBV, but damn severe side effects like severe taste in your mouth all the time and anal itching AND specific to genotype 1 yet 6 Dif genotypes out there, like inEgypt it’s 4 so would not be useful for them (and highest burden there)
ALTHOUGH 50-70% SVR - HCV is highly mutable, viral resistance can quickly emerge if given alone (after 3 days only) so combination therapy is really a must
10
Q
What are the two types of polymerase inhibitors?
A
- NS5B inhibitors can either be nucleoside analogues (bind into active site since elk like nucleotide) or can be non-nucleoside analogues (bind to allosteric sites)
11
Q
Example of polymerase inhibitor and how it works?
A
- Sofosbuvir (FDA approved in 2013)
- nucleoside inhibitor (U analog)
- Mechanism: chain termination cuz cannot add other nucleotide after sofosbuvir
- 12 week duration (daily for 12 weeks): pan genotypic
- and over 90% SVR for genotype 1, about 82% SVR for genotype 4, very good in combination
- BUT COST: 80K/course of treatment though today about 20K/course in Canada
- It is a pro-drug so is a nucleoside and then cellular enzymes transform it into monophosphate form all the way till triphosphate
12
Q
What about the level of resistance to sofosbuvir?
A
In mono therapy experiment only 1 in 1,000s of patient showed resistance, so it IS quite rare
13
Q
About NS5A inhibitors: discovery?
A
NS5A: phosphoprotein, role in antiviral response inhibition, RNA replication (rep organelle biogenesis and interacts with 3’ end of viral RNA as well as NS5B) and assembly it carries RNA for replication organelle to assembly site
Hard to screen for drugs for this protein because no enzymatic activity, instead using stable replicon and screening compound libraries and found drug that worked although didn’t know target, grew replicon cells at very low levels of drug to check for resistant strains and then sequenced the resistant viruses and found that all mutation in NS5A, so must be target
14
Q
NS5A domains?
A
- amphipathic helical to tether to ER membrane
- Domain1 RNA binding domain
- other two domains are intrinsically disordered so not shown
15
Q
What is the proposed mechanism of action?
A
- think that inhibitor binds to bottom of domain 1 and traps the protein in conformation where RNA not able to fit in the channel (dimer channel binds RNA), maybe protein-protein interactions?
- But we know it does bind the dimer, because of the specificity for dimer interface pocket and has two fold symmetry which helps interact with both monomers of the dimer
- however only know target definitely not known exact mechanism
16
Q
What are the two NS5A inhibitors?
A
- Daclatasvir and Ledipasvir (FDA 2014)
- 12 week duration, pan genotypic
- combination therapy of DAAs since enough now and has less side effects since no non specific IFN and RBV but IFN and RBV free
- Higher efficiency, shorter duration
- 94% SVR for genotype 1
- high barrier to resistance in combination
- Cost is about 80K/course
- Many other drugs are in clinical trials most of which are DAAs of the three classes discussed
17
Q
What are the implications of the diversity of HCV?
A
- Implications for immune response, disease progression, and treatment (hello drug resistance)
18
Q
What about the mutability of HCV dictates drug resistance?
A
- Theoretically, every possible single and double mutant in the HCV RNA genome can be produced every single day in every patient because of the viral turnover, error pol rate and the different base variants
19
Q
What does mutability of HCV mean for infection?
A
- HIV does not really exist as a single virus, exists more as a mixture of populations of genetically distinct, but closely related (each about 2 mutations), virions in ever patient (quasi species)
- Most resistant viruses are relatively unfit (worse at replicating, and won’t be selected over time) and are undetectable prior to therapy (not the dominant variants since unfit although probably exist in the patient)
20
Q
What are the kinetics of viral drug resistance?
A
- Drug treatment gets rid of WT and drug susceptible BUT drug resistant variant will accumulate and this is why we never use mono therapy in hopes that will not be resistant to all treatments unlikely resistant to all treatments because this wouldn’t really be a fit virus
21
Q
What factors determine the treatment response when it comes to maximizing response and minimizing resistance?
A
- Virus: the genetic barrier is related to the number and type of mutations require to overcome the clinical activity of a regimen (mutations that decrease the viral fitness increase resistance barrier)
- Drugs: the pharmacological barrier is increased by higher potency and high drug levels
- patient: tolerability of a regimen as well as patient adherence are critical to treatment success
22
Q
So back to that one patient that showed resistance to sofosbuvir?
A
- After treatment, virus came on strong and when sequenced showed that 100% of virus had a mutation in the S282T active site of the polymerase but this makes it harder to incorporate normal nucleotides, virus is pretty unfit and eventually when sequenced later, wild type had returned, so then they attempted a treatment of RBV and sofosbuvir
- Sofosbuvir was good for the wild-type version and RBV is smaller than a normal nucleotide so it could be incorporated after S282T mutation and can attack this strain so the combination was able to cure the patient
23
Q
So what are the general conclusions of the treatment of HCV?
A
- HCV is curable but resistance can emerge and the high cost of treatment means that accessibility is a barrier in many places where the burden is highest BUT state if emergency in Egypt and India allowed them to get the drug for much cheaper
- HCV exists as a quasi species so resistant variants exist way before treatment and they can be selected for during treatment, drug resistance can occur during treatment with any or all antiviral drugs, resistance is a consequence of treatment failure and not always the cause
24
Q
Perspectives for new and future treatments
A
We will always be aiming for perfectovir, maximize tolerability and efficacy, think about now with new HCV treatments much shorter duration of 12 weeks and all oral no painful jabs
25
How can we plan to eliminate viral hepatitis by 2030?
- Need a national action plan
- Political Will
- Strong public health system in place
26
What is the HCV cascade of care?
- about 3.5 million burden of HCV in USA, only 50% are diagnosed and aware, 43% have access to outpatient care, 27% have confirmed that they are HCV RNA positive, 17% got the liver biopsy, 16% got treatment prescribed and only 9% have achieved SVR
27
What is the trend of deaths for infectious diseases?
- hepatitis deaths are still trending up despite other infections trending down (HIV, malaria, TB)
28
What are some barriers in Canada for achieving a hepatitis free world?
- access to care and retention, need more education with family doctors who can maybe offer longterm follow up that is not always possible with specialists, must have stage 2 fibrosis before access to medication, cost of treatment, must advocate on behalf of drug users, need better diagnosis like birth cohort screening, enhanced testing
- Lack of government funding and political will, healthcare delivery in Canada, need public health advocacy, need competition and alternative drug pricing strategies
- Modelling suggests that increasing uptake to over 10,800 patients a year (3X current practice) over the next 20 years could help realize the HCV elimination in Canada
29
What other ways could we target a virus without targeting THE virus?
Targeting host-virus interactions
Advantages: every stage of life cycle is highly dependent on the hosts, high barrier to resistance because virus is mutable but not us lmao, pan-genotypic activity if choose something needed by all genotypes, wide range of possible druggable-targets (since viral targets are limited with only like 10 possible proteins for example)
However, efforts to develop this kind of drug are hampered by toxicity, lack of predictive culture models or complex nature of host-virus interactions
30
What are microRNAs?
- small non coding RNA
- bind to partially complementary sites (seed sequences) in the 3' end of mRNAs and block their translation
- Typically turns OFF gene expression
- expressed in a tissue-specific manner
- Regulates 50-60% of all human genes
31
About miR-122?
Evolutionarily conserved and liver specific microRNA (even in zebrafish)
- 70% of microRNAs in the liver between 66k and 132k copies per cell
- regulates cholesterol synthesis and fatty acid metabolism
32
How is the miR-122 interaction with HCV unusual?
- 5' end though usually only 3' end
- 2 binding sites right next to each other at terminal base of HCV
- up regulates virus instead of downregulating gene expression
33
How can miR-122 sequestration help treat HCV?
- it promotes the HCV RNA accumulation
- so sequestration abolishes accumulation dn this has been observed in patitent, chimpanzees and cell culture
34
What is the mechanism of action of miR-122 with HCV genome?
- acts as a cap to protect the HCV genome from degradation by pyrophosphatases and other nucleases as they cannot access the 5' terminus anymore, it also promotes translation, and acts as an RNA chaperone or riboswitch
- Site 1 is base paired and site 2 is free so it gets bound first which then causes a conformational switch and site 1 is free to be bound by another protein (riboswitch) and this leads to preotection of the 5' end (stability), the two proteins are now so close together so the second one shifts a little bit and sticks elbow into the IRES to stabilize it (better translation)
35
What is the suggested course of treatment using an antisense miR-122 inhibitor?
- miR-122 sites are conserved across all HCV genotypes, in fact all hepaciviruses may be rued by that one microRNA so one single-dose administration of RG-101 can be used
- mean viral load reduction of 4.1 logs and 6 out of 14 patients showed RNA levels below detectable and 2 with a single does
- Pan genotypic activity
- Side effects => lowers cholesterol
- unique mechanism of targeting a host factor
36
What was a recent event indicating expansion of the hepacivirus genus?
- until 2011, origin a mystery
- but then an outbreak of a respiratory disease in dogs lead to the identification of a novel hepacivirus Canine hepacivirus (CHV)
37
After dogs, what else was seen?
- Using the CHV NS3 antigen, screened numerous mammalian species and found that 35% of horses were seropositive and renamed CHV to NPHV, so horses became a source of tentative evidence for etiologic role of NPHV in hepatitis
- And then using other viral discovery tools like RNA seq, found tons of hepaciviruses in tons of species, rodents, bats, money, cows and even sharks (1st non-mammalian host)
- perhaps evidence for a zoonotic origin of HCV, similar to vector-borne flaviviruses and pestiviruses
38
What are the hypotheses regarding the origin of HCV?
- 1: single cross-species transmission event between ancestral humans and zoonotic host which is reflected in geographic populations and the global patterns of HCV diversity
- 2: multiple independent zoonotic transmission events, for example rodent hepaciviruse are the most genetically divers (3 lineages and 8 clades), common ancestors with other animal heapciviruses so could an intermediate or original host
39
Conclusion of HCV origin?
- hepaciviruses may have infected different animals including humans throughout evolution or even before host speciation with high species specificity, propensity for persistence and long-term coevolution and host adaptation
- Plausible that like other flaviruses, some hepaciviruses could be transmissible via and insect vector or airborne route as respiratory swabs with CHV and arthropod sampling
